It was recently estimated that between the years 1998 and 2030 there will be 225 million new cases of tuberculosis (TB) and 79 million TB-related deaths (Murray, C. J. L., and J. A. Salomon, 1998). These numbers are astonishing considering that treatments for this disease, in the forms of vaccines or chemotherapy, have been available for more than 50 years (Kramnik, I., et al., 2000). Mycobacterium tuberculosis, the causative agent of TB, is notoriously slow-growing and, during infection, can persist in a latent form in many individuals. These attributes contribute to the reasons why typical chemotherapy regimens for TB last 6-9 months (Bloom, B. R., and J. D. McKinney, 1999) and why TB is so persistent. This prolonged treatment presents significant hurdles in developing new antibiotics and in retaining the efficacy of currently used antibiotics. Side effects and toxicity from a particular compound can be magnified when a patient takes a drug for this length of time, and there are increased incidences of poor adherence to the chemotherapy regimen by unmonitored patients, resulting in the development of multidrug-resistant TB (MDR-TB) infections. These facts, together with alarming interactions between HIV and TB infections that can result in increased numbers of infected individuals and MDR-TB (Dye, C., et al., 2002; Gupta, R., et al., 2001; Lawn, S. D., et al., 2002; Barnes, P. F., et al., 1991; Bloom, B. R., and C. J. Murray, 1992), make it of paramount importance to develop new chemotherapy agents or introduce modifications to current agents to reduce toxicity and increase activity against MDR-TB.
One of the earliest antibiotics developed for the treatment of a bacterial infection was streptomycin, and its initial (and continued) use was for the treatment of tuberculosis (TB), the clinical manifestation of a M. tuberculosis infection. For over 50 years additional antibiotics and a vaccine have been developed to fight this infection. However, despite these developments this organism remains a significant human health concern.
Currently, the successful treatment of TB typically requires the simultaneous use of at least three drugs. These drugs are grouped into “first-line” and “second-line” antibiotics. The first-line drugs (e.g. streptomycin) are used first because they are less toxic and less expensive. It has been recommended that the second-line drugs (including viomycin, tuberactinomycins and capreomycins) be reserved specifically for the treatment of MDR-TB (Croft, J., et al., 1997). Use of the second-line drugs is increasing because resistance to many, if not all, of first-line drugs is increasing (Frieden, T. R., et al., 1993; Goble, M., et al., 1993), and it has been proposed that MDR-TB will soon be the norm (Davies, J. 1996). It has, however, been more than 25 years since a new drug to combat TB has been introduced (Duncan, K., and J. C. Sacchettini, 2000). Thus there is a continuing need in the art for the identification of antibiotics useful for the treatment of TB and particularly MDR-TB.
The tuberactinomycin family (TUBs) of antibiotics, including viomycin (VIO), tuberactinomycins (TUBs), capreomycins (CAPs) and tuberactinamines (FIG. 1) are a family of cyclic peptide natural products that are important second-line antibiotics for the treatment of MDR-TB. In fact, certain TUBs are included on the World Health Organization's “List of Essential Medicines” because of their anti-MDR-TB activity (WHO, 2002). Initial interest in the tuberactinomycins stemmed from the observation that VIO, the first tuberactinomycin to be isolated (Bartz, Q. R., et al., 1951; Ehrlich, J., et al., 1951; Finlay, A. C., et al., 1951), had the unusual property of having higher antimicrobial activity against mycobacterial species than against other bacteria (Ehrlich, J., et al., 1951; Finlay, A. C., et al., 1951; Marsh, W. S., et al., 1953 U.S. Pat No. 2,633,445; Mayer, R. L., et al., 1954). Importantly, VIO was active against strains of M. tuberculosis that were resistant to streptomycin (Hobby, G. L., et al., 1953). More recently, TUBs (Nagata, A., et al., 1968) and CAPs (Herr, E. B. J., et al., 1962 U.S. Pat. No. 3,143,168) were found to share a similar spectrum of antimicrobial activity with VIO. Currently, TUB N (FIG. 1) is used in Asia for the treatment of M. tuberculosis (Tsukamura, M., et al., 1989) and M. avium complex (Shigeto, E., et al., 2001) infections, while the CAPs are used in combination with other anti-TB drugs to treat MDR-TB (Goble, M. 1994).
New TUB derivatives are needed to combat the ever-expanding mycobacterial resistance to these drugs. A recent study analyzing 46 different strains of M. tuberculosis from TB patients found that 10% of these strains were resistant to CAP (Fattorini, L., et al., 1999). The continued spread of resistance without the development of new therapeutic alternatives will be devastating for patients who have limited options for treatment. It has recently been reported that 15 of 158 TB patients required treatment with CAP because the use of the standard aminoglycosides (amikacin, kanamycin, or streptomycin) was not appropriate (Tahaoglu, K., et al., 2001). Without the option of CAP, these patients would face an uncertain future.
In addition to their historical use in treating TB, the TUBs and analogs thereof have become lead compounds for use in treating other bacterial infections such as vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (Dirlam, J. P., et al., 1997; Linde II, R. G., et al., 1997; Lyssikatos, J. P., et al., 1997), and in targeting catalytic RNAs to disrupt viral replication (Jenne, A., et al., 2001; Rogers, J., et al., 1996; Schroeder, R., et al., 2000; von Ahsen, U., et al., 1991; Wank, H., et al., 1994; Wank, H., and R. Schroeder, 1996). In these cases, the TUBs are also considered important as the starting compounds for the development of more potent drugs.
Tuberactinomycins are reported to inhibit group I intron RNA splicing at high concentrations (Wank, H., et al., 1994). At subinhibitory concentrations, they are reported to stimulate oligomerization of group I intron RNA and intermolecular reactions (Wank, H., and R. Schroeder, 1996). The former finding is of interest for targeting group I introns in pathogenic microorganisms, since this type of intron is not found in humans (Hermann, T., and E. Westhof, 1998). The latter finding is of interest for developing therapeutic ribozymes that can fix mutated RNAs involved in inherited diseases (James, H. A., and I. Gibson, 1998).
TUBs are also reported to inhibit the human hepatitis delta virus ribozyme (Rogers, J., et al., 1996), and recently it was reported that VIO binds to subdomains Ille/f of the hepatitis C virus (HCV) internal ribosome entry site, blocking HCV translation (Vos, S., et al., 2002). These studies indicate that derivatives of tuberactinomycins will be useful as antiviral agents.
Recent studies using TUB derivatives for the treatment of infections by the animal pathogen Pasteurella haemolytica and the human pathogens vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (Dirlam, J. P., et al., 1997; Linde II, R. G., et al., 1997; Lyssikatos, J. P., et al., 1997) found that modifications to the cyclic pentapeptide core of TUBs could enhance their activity against these pathogens. This work extends earlier findings that chemical modifications of these antibiotics can extend their use to non-mycobacterial bacteria (Kitagawa, T., et al., 1979, 1976, 1975; Wakamiya et al., 1977). Thus, new TUB derivatives are likely candidate drugs for the treatment of other bacterial infections.
New antibiotics and variants or derivatives of known antibiotics can be obtained by screening of natural sources, by manipulation of biosynthetic pathways in antibiotic producing organisms or by a combination of biosynthesis and chemical synthesis.
Gene Clusters
The study of the biosynthesis of natural products has made significant advancements in recent years due to the understanding that bacteria cluster the genes encoding all the enzymes involved in the biosynthesis of a particular natural product into one region of its genome (Chater, K. F., and C. J. Bruton. 1985; Du, L., et al., 2000; van Wageningen, A. M. A., et al., 1998). Analysis of this sequence allows a researcher to develop testable models for how the necessary precursors are synthesized, condensed, and modified to generate the final metabolite. In addition to the basic understanding of how a compound is biosynthesized, this information has the ability to direct metabolic engineering of the pathway to generate previously unattainable structural diversity in the metabolite of interest. This approach can be used to transform the developmental process of new pharmaceutically and agriculturally important compounds.
Chater and Bruton (1985) recognized that genes conferring resistance to, as well as controlling regulation and production of, methylenomycin are clustered in S. violaceus-ruber and S. coelicolor. The close linkage between the gene conferring resistance to the antibiotic and the other genes involved in biosynthesis of the antibiotic provides the basis for isolating an antibiotic cluster if the gene conferring antibiotic resistance is known.
Subsequent studies have confirmed that genes that confer resistance to an antibiotic and genes involved in the biosynthesis of that antibiotic, including penicillin, cephalosporin and cephamycins, and associated secondary metabolites, are organized in clusters. (Martin, 1992; See review by Martin and Liras, 1989). These biosynthetic clusters typically contain at least one pathway-specific regulatory gene and at least one resistance gene. U.S. Pat. No. 4,935,340 (Baltz et al., Method of Isolating Antibiotic Biosynthetic Genes, 1990) reports a method for identifying and isolating an antibiotic biosynthetic gene via hybridization with a labeled antibiotic resistance-conferring gene. This method relies on the fact that the majority of antibiotic biosynthetic genes from antibiotic-producing organisms are linked to antibiotic resistance-conferring genes. In particular, Baltz et al. used the erythromycin resistance-conferring gene to identify erythromycin biosynthetic genes via their hybridization method. In addition, they identified a recombinant vector that encoded erythromycin biosynthesis to drive erythromycin expression in a host (Streptomyces lividans TK23) that when untransformed produced no measurable amount of erythromycin.
A biosynthetic gene cluster for vancomycin group antibiotics was identified from Amycolatopsis orientalis (van Wageningen et al. 1997). In particular, 39 putative genes spanning 72 kb of contiguous DNA, including genes encoding for chloroeremomycin biosynthesis, were identified. Other antibiotic gene clusters that have been identified include those for rifamycin (August et al., 1998. Chem Biol. 5:69-70), tetracenomycin (Guilfoile & Hutchinson, 1992, Journal of Bacteriology, 174: 3651 & 3659) and actinorhodin (Caballero et al, 1991, Mol Gen Genet., 228: 372-80).
The mitomycin biosynthetic gene cluster was recently isolated and characterized from S. iavendulae (Sherman et al., U.S. Pat No. 6,495,348). The mitomycin gene cluster contains 47 mitomycin biosynthetic genes spanning 55 kb of contiguous DNA. These genes include those which encode for polypeptides which function to generate precursor molecules, such as those for mitosane ring system assembly, and those to functionalize sites on the core mitosane ring system. U.S. Pat. No. 6,495,348 and others (see e.g. Chater; U.S. Pat. No. 4,935,340), report that genes that encode enzymes for natural product assembly, including antibiotic production, are clustered on the Streptomyces genome. Furthermore, genes associated with antibiotic resistance (mrt and mrd) were located within the mitomycin gene cluster. This is consistent with previous studies that indicated antibiotic biosynthetic gene clusters typically contain one or more genes that confer antibiotic protection (Seno and Baltz, 1989).
By disrupting a repressor gene, mitomycin production in S. Iavendulae is reported to increase several-fold (U.S. Pat No. 6,495,348). E. coli were transformed to co-express MRD and MCT, the mitomycin-resistance conferring proteins, so that transformed cells had a high level of resistance to mitomycin. This resistance was mediated by increased mitomycin export out of the cell. Thus, as in Baltz et al., the use of antibiotic biosynthetic clusters in expression cassettes can be used to drive expression of antibiotics in host cells that normally do not produce measurable quantities of the antibiotic, and to increase the production and yield for cells that normally produce the antibiotic.
Organisms that do not naturally produce a particular biological product can be transformed with biosynthetic genes to produce that biological product. This is exemplified in U.S. Pat. No. 6,391,583 (Hutchinson et al., Method of Producing Antihypercholesterolemic Agents, 2002), where increased production of a cholesterol lowering compound, lovastatin, in both lovastatin-producing and non-lovastatin-producing producing organisms, was disclosed using a cluster of 17 genes from a native-lovastatin-producing strain of bacteria (A. terreus). By inactivating certain genes contained within the lovastatin cluster, different HMG-CoA reductase inhibitors were generated in the host organism. By mutating certain genes it was possible to prevent lovastatin production. By introducing extra copies of other genes into A. terreus, it was possible to increase lovastatin production up to 7-fold. Introducing the entire lovastatin-cluster into a normally non-lovastatin producing cell can result in lovastatin production in the cell.
These studies show that it is well known in the art to use gene clusters to affect production of a biologically active product, including increasing production in a native producer, abolishing production of the biologically active product, and forcing production of the biologically active product in a host cell that normally does not produce the biologically active product. It is also known that by selectively inactivating certain genes by mutation, or transforming a host cell with only certain genes, it is possible to selectively generate particular precursors of the biologically active product, which themselves can be biologically active, and to generate novel derivatives of these precursors. In addition, directed biosynthesis wherein an alternative precursor is applied to these transformed cells can be utilized to manufacture novel antibiotics.
Thus, there is a continuing need in the art for identification and isolation of antibiotic biosynthetic genes, including genes that result in enhanced production of antibiotics and confer resistance to antibiotics. Understanding the antibiotic's biosynthetic pathway also allows novel antibiotics to be manufactured biosynthetically.
Chemical Variants
It is also known in the art that individual precursors of antibiotics can be isolated and purified from a transformed cell culture, and chemically modified to generate novel derivatives thereof. This is a semi-synthetic method of synthesis. In addition, it is well known in the art that altering fermentation conditions can alter antibiotic production and provide useful starting points for the production of new semi-synthetic antibiotics. Gastaldo L, and Marinelli F. Microbiology. 2003 June; 149(Pt 6):1523-32.
Such techniques involve a combination of biosynthetic and chemical techniques. For instance, it can be difficult to manufacture antibiotics solely by chemical means. However, isolating a precursor molecule produced biosynthetically in an organism permits the generation of novel analogs by chemical means. For example, Dirlam et al. (1997) modified a synthetic reaction reported by Momoto and Shiba (1977) that used ureido exchange reactions on tuberactinomycin N. 6a-(3′,4′-dichlorophenylamino)capreomycin was prepared by treating capreomycin sulfate with a 40-fold excess of 3,4-dichloroaniline in 2 N HCl/dioxane at 65° C. for 4 hours. Phenyl urea analogs could be generated in a similar manner. Other analogs were generated by reduction of the C-6-C-6a double bond by use of triethylsilane in trifluoroacetic acid. The activity of these derivatives was measured by assaying for antibacterial activity in different bacteria.
In addition to C-6a aryl urea modification, C-19 modification to viomycin and β-lysine substitutions and modification by chemical means have been reported. Lyssikatos et al., 1997; Linde et al., 1997. Such chemical modification studies were conducted in the hope of identifying antibiotic derivatives with improved potency. The free amino groups of the β-lysine residue in viomycin have also been chemically modified (Kitagawa et al., 1976) in an effort to determine the importance of the β-lysine residue in VIO's antimicrobial activity. Wakamiya et al. (1977) disclosed the antimicrobial activity for various TUB analogs where different amino acids were attached to the free α-amino group of the α,β-diaminopropionic acid residue in TUB N.
The ability to chemically generate antibiotic derivatives is limited by the amount, variety and purity of the starting material. Thus, need in the art remains for the generation of novel, chemically-pure antibiotic derivatives to serve as templates for chemical modification to generate improved antibiotics.